Detection of Trypanosoma cruzi and Trypanosoma rangeli Infection by Duplex PCR Assay Based on Telomeric Sequences

We used the species specificity and repetitious nature of subtelomeric kinetoplastida sequences to generate a duplex PCR assay for the simultaneous detection of Trypanosoma cruzi and Trypanosoma rangeli in experimentally and naturally infected triatomine (Reduviid) bugs and in infected human subjects. The assay was species specific and was capable of detecting 1/20th of T. cruzi and 1/4th of T. rangeli cell equivalents without complementary hybridization. In addition, the PCR-based assay was robust enough for direct application to difficult biological samples such as Reduviid feces or guts and was capable of recognizing all T. cruzi and T. rangeli strains and lineages. Because the assay primers amplify entirely different target sequences, no reaction interference was observed, facilitating future adaptation of this assay to an automated format.     

Expression of fluorescent genes in Trypanosoma cruzi and Trypanosoma rangeli (Kinetoplastida: Trypanosomatidae): its application to parasite-vector biology

Two Trypanosoma cruzi-derived cloning vectors, pTREX-n and pBs:CalB1/CUB01, were used to drive the expression of green fluorescent protein (GFP) and DsRed in Trypanosoma rangeli Tejera, 1920, and Trypanosoma cruzi Chagas, 1909, isolates, respectively. Regardless of the species, group, or strain, parasites harboring the transfected constructs as either episomes or stable chromosomal integrations showed high-level expression of fluorescent proteins. Tagged flagellates of both species were used to experimentally infect Rhodnius prolixus Stal, 1953. In infected bugs, single or mixed infections of T. cruzi and T. rangeli displayed the typical cycle of each species, with no apparent interspecies interactions. In addition, infection of kidney monkey cells (LLC-MK2) with GFP-T. cruzi showed that the parasite retained its fluorescent tag while carrying out its life cycle within cultured cells. The use of GFP-tagged parasites as a tool for biological studies in experimental hosts is discussed, as is the application of this method for copopulation studies of same-host parasites.     

Serological Diagnosis of Trypanosoma rangeli Infected Patients. A Comparison of Different Methods and its Implications for the Diagnosis of Chagas' Disease

Venous blood from 65 Panamanian schoolchildren living in an area endemic for both Trypanosoma cruzi and T. rangeli were screened for the presence of these parasites. Trypanosoma rangeli were isolated and cultured from four individuals. Serological tests of all 65 sera were performed, including immunohaemagglutination (IHA), indirect immunofluorescence assay (IF) and ELISA using both T. rangeli and T. cruzi as antigens, as well as T. cruzi synthetic peptides in an ELISA assay. Results indicated a higher immunoreactivity to T. rangeli preparations than to T. cruzi within the studied population, which could be divided into four 'serological responder' groups. Interestingly, the panel of SAPA and other T. cruzi synthetic peptides were not useful in the discrimination of patients. Furthermore, patients from whom parasites had been isolated could not be distinguished from those of two other groups. Significant immunoreactivity to T. cruzi preparations was displayed in all responder sera, despite total lack of evidence of infection with this parasite. The immunobiological significance of T. rangeli infection is unclear, but these data indicate that it is a compounding problem in the accurate diagnosis of pathological T. cruzi infection by serological analysis. The relationship of these cohabiting species, in respect to infection outcome and immunological activation, is discussed.     

trans-Sialidase neutralizing antibody detection in Trypanosoma cruzi-infected domestic reservoirs

The detection of Trypanosoma cruzi infection in domestic dogs and cats is relevant to evaluating human transmission risks and the effectiveness of insecticide spraying campaigns. However, the serological assays routinely used are associated with cross-reactivity in sera from mammals infected with Leishmania spp. We used a trans-sialidase inhibition assay (TIA) for T. cruzi diagnosis in serum samples from 199 dogs and 57 cats from areas where these types of infections are endemic. TIA is based on the antibody neutralization of recombinant trans-sialidase, an enzyme that is not detected in the coendemic Leishmania species or Trypanosoma rangeli parasites. T. cruzi infection was also evaluated by conventional serology (CS) (indirect immunofluorescence, indirect hemagglutination, enzyme-linked immunosorbent assay, and immunochromatographic dipstick test) and xenodiagnosis. Sera from 30 dogs and 15 cats from areas where these organisms are not endemic and 5 dogs with visceral leishmaniasis were found to be nonreactive by TIA and CS. Samples from dogs and cats demonstrated 91 and 95% copositivities between TIA and CS, whereas the conegativities were 98 and 97%, respectively. Sera from xenodiagnosis-positive dogs and cats also reacted by TIA (copositivities of 97 and 83%, respectively). TIA was reactive in three CS-negative samples and was able to resolve results in two cat serum samples that were CS inconclusive. Our study is the first to describe the development of trans-sialidase neutralizing antibodies in naturally infected dogs and cats. High CS conegativity and the absence of trans-sialidase neutralization in dog sera from areas where leishmaniasis is not endemic and from dogs with visceral leishmaniasis support TIA specificity. The TIA may be a useful tool for T. cruzi detection in the main domestic reservoirs.     

The evolution of two Trypanosoma cruzi subgroups inferred from rRNA genes can be correlated with the interchange of American mammalian faunas in the Cenozoic and has implications to pathogenicity and host specificity

The agent of Chagas disease, Trypanosoma cruzi, is divided into two highly divergent genetic subgroups, lineages 1 and 2, which include all typed strains isolated from humans, insect vectors, and sylvatic mammals. The evolutionary origin of these two T. cruzi lineages and the clinical importance of their identification, have been the subject of intense debate. Here, using molecular phylogenetic analysis, we found that the distance between the two T. cruzi lineages is equivalent to the distance between genera Leishmania and Endotrypanum. Also, we confirmed that T. rangeli is more closely related to T. cruzi than to T. brucei using the rDNA sequence from a human strain of T. rangeli. Phylogenetic trees based on small subunit rDNA sequences further suggest that the two T. cruzi lineages diverged between 88 and 37 million years (Myr) ago. We hypothesize that lineage 2 is indigenous to South America while lineage 1 has been introduced to South America recently, along with North American placental mammals, after the connection of the Americas in the Pliocene (5 Myr ago) or with caviomorph rodents and primates in the Oligocene (38 Myr ago). This would explain the preferential association of T. cruzi lineage 2 with marsupials and of lineage 1 with human disease. These two T. cruzi lineages are likely to be distinct species, or at least subspecies, because of their different ecological and epidemiological traits and estimated long period of independent evolution.     

Radiolabeled total parasite DNA probe specifically detects Trypanosoma cruzi in mammalian blood.

A DNA-DNA hybridization procedure is described in which radiolabeled total parasite DNA is used to detect Trypanosoma cruzi spotted directly onto nylon membrane. Under conditions that favor hybridization of repetitive DNA sequences, the radiolabeled total DNA was able to detect as few as five parasites spotted onto nylon membrane. Trypanosomes were detectable in blood from mice with T. cruzi parasitemias spotted directly onto membranes. All 10 strains of T. cruzi examined, which came from different areas of the United States and South America, were readily detected with a total DNA probe from any 1 strain of the parasite. No signals were detected with up to 20,000 Trypanosoma rangeli, T. brucei, Crithidia fasciculata, or Leishmania parasites or with normal mouse blood or mammalian DNA. The hybridization method is sensitive, specific, rapid, and inexpensive and is potentially applicable to the detection of other parasites.     

Immunoparasitological Studies of Trypanosoma Cruzi Low Virulence Clones from Panama: Humoral Immune Responses and Antigenic Cross-Reactions with Trypanosoma Rangeli in Experimentally Infected Mice

The kinetics of humoral immune responses were investigated in mice experimentally infected with five clones of Trypanosoma cruzi isolated from different sources in Panama. Sera were collected at different timepoints post-infection. ELISA and IHA tests were used to detect antibodies against T. cruzi epimastigote antigens. The levels of T. cruzi specific antibodies increased during the course of infection; at day 90 post-infection the range was between 1:5120 and 1: 10240. A high correlation was evident between ELISA and IHA results. Western blots revealed that these antibodies recognized polypeptides of 81, 76 and 71 KDa during the first weeks and 81, 76, 71, 50, 40, 28 and 12 KDa after 30–50 days. Only minor differences in antigen recognition patterns were demonstrated, suggesting that the major antigens may be represented in all clones. T. rangeli antigens were also recognized by T. cruzi seropositive sera. However, an ELISA test using antigens isolated from a genomic expression library of T. cruzi revealed that a hyperimmune rabbit serum against T. rangeli was unable to recognize the repeat sequence of SAPA (Shed Acute Phase Antigen) peptides but did recognize a number of other T. cruzi synthetic peptide antigens. The importance of these findings, in the context of Chagas' disease, is discussed.     

Control of Trypanosoma cruzi Infection in Mice Deprived of T-Cell Help

The role of CD4+ T lymphocytes in the resistance of BALB/c mice to Trypanosoma cruzi was examined by in vivo depletion using monoclonal anti-CD4 antibodies (MoAbs). When the administration of MoAbs was initiated 2 days before, or 5 to 12 days after the infection (dpi) with 50 bloodstream-form trypomastigotes of the Tulahuén strain, mice showed an enhanced susceptibility to the parasite. Specific IgG, but not IgM responses, were inhibited in anti-CD4-treated and infected mice. However, when anti-CD4 treatment of mice was delayed until the 8th week of infection, neither a reactivation of the infection as determined by mortality or parasitaemia, nor a modulation of the titre of anti-T. cruzi IgG antibodies was detected. Furthermore, mice chronically infected with T. cruzi and deprived of CD4+ T cells resisted the challenge with 50,000 trypomastigotes (approximately 1000 LD50). Secondary antibody responses against parasite antigens were inhibited after in vitro depletion of CD4+ cells in chronically infected mice before boosting with T. cruzi antigens. However, recipients of CD4 or T-cell-depleted spleen cells from mice chronically infected with T. cruzi were protected when challenged with the parasite. The possibility that the parasite control is maintained by long-lived B cells capable of rapid differentiation into IgG-secreting plasma cells in the absence of T helper cells is discussed considering the present data.     

Identification of six Trypanosoma cruzi lineages by sequence-characterised amplified region markers

Six discrete phylogenetic lineages were recently identified in Trypanosoma cruzi, on the basis of multilocus enzyme electrophoresis and random amplified polymorphic DNA (RAPD) characterisation. The objective of the present study was to develop specific PCR-based markers for the identification of each of the six lineages. Eighty-seven T. cruzi stocks representative of all the lineages were characterised by RAPD with three primers, resulting in the identification of three fragments that were specifically amplified in the given sets of lineages. After cloning and sequencing these fragments, three pairs of sequence-characterised amplified region (SCAR) primers were designed. After PCR amplification using the SCAR primers, the initial polymorphism was retained either as the presence or absence of amplification, or as size variation between the PCR products. Although most PCR products, taken individually, were distributed across several lineages, the combination of the three SCAR markers resulted in characteristic patterns that were distinct in the six lineages. Furthermore, T. cruzi lineages were distinguished from Trypanosoma rangeli, T. cruzi marinkellei and T. cruzi-like organisms. The excellent correspondence of these new PCR markers with the phylogenetic lineages, allied with their sensitivity, makes them reliable tools for lineage identification and strain characterisation in T. cruzi. The approach described here could be generalised to any species of microorganism harbouring clear-cut phylogenetic subdivisions.     

Restriction fragment length polymorphisms in the ribosomal gene spacers of Trypanosoma cruzi and Trypanosoma conorhini

The ribosomal RNA genes of two species of Trypanosoma, Trypanosoma cruzi, the etiological agent of Chagas' disease, and Trypanosoma conorhini, a non-pathogenic rodent trypanosome, were cloned and partially characterized. The physical maps derived for their rRNA genes were similar throughout the region that encompasses the SSU-and LSU-rRNA coding sequences. However, the non-transcribed spacer DNA of both T. cruzi and T. conorhini was found to be polymorphic for several restriction enzyme sites. We show that strains of T. cruzi can be typed according to the characteristic restriction fragment length polymorphism of their NTS DNAs.     

Specific binding of human plasma high density lipoprotein (cruzin) to Trypanosoma cruzi

Binding of high density lipoprotein (HDL) to Trypanosoma cruzi was examined because of its ability to specifically inhibit the parasite's neuraminidase. 125I-Labeled HDL bound to live and glutaraldehyde-fixed parasites equally well either at 37 degrees C or at 4 degrees C. Binding was saturable and inhibited by unlabeled HDL but not by unrelated plasma proteins. Specificity of the T. cruzi-HDL interaction was confirmed using fluorescein labeled HDL which bound to T. cruzi but not to T. rangeli, a species whose neuraminidase is not inhibited by HDL. Binding of HDL to T. cruzi paralleled the neuraminidase activity exhibited by the parasite's different stages and strains. In agreement with this finding, Steck and Wallach analysis of the binding data showed that the number of HDL binding sites was greater in infective trypomastigotes and on strains with high neuraminidase activity. However, the association constant of the binding did not change within the various developmental forms and strains of T. cruzi, suggesting that HDL bound to the same receptor, presumably having neuraminidase activity.     

Characterization of tubulin genes in Trypanosoma rangeli

Tubulin genes in Trypanosoma rangeli, the only trypanosome besides T. cruzi to infect humans in America, are organized in homogeneous, alternate alpha and beta gene tandem repeats of 3.8 kb. The basic repeat was cloned, mapped and partially sequenced. In contrast to most other eukaryotes, where tubulin genes are scattered throughout the genome, trypanosomatids so far studied are characterized by tandem arrangements of these genes with the genus Trypanosoma displaying an alternating alpha- and beta-tubulin tandem repeat.     

Limit of detection of PCR/RFLP analysis of cytochrome oxidase II for the identification of genetic groups of Trypanosoma cruzi and Trypanosoma rangeli in biological material from vertebrate hosts

Parasitology Research - Mixed infections with Trypanosoma cruzi and Trypanosoma rangeli and their different genetic groups occur frequently in vertebrate hosts and are difficult to detect by...     

Trypanosoma rangeli Tejera, 1920, in chronic Chagas’ disease patients under ambulatory care at the Evandro Chagas Clinical Research Institute (IPEC—Fiocruz, Brazil)

We report the finding, the isolation by hemoculture, and the characterization of Trypanosoma rangeli stocks from two chronic Chagas' disease patients who received ambulatory care at the Evandro Chagas Clinical Research Institute (IPEC, FIOCRUZ). Both patients proceeded from Bahia State (Brazil). One of them presented the cardiac form of the disease and the other indeterminate symptomalogy. Giemsa-stained smears of the hemocultures from these patients evidenced that they were coinfected with T. rangeli and Trypanosoma cruzi, with predominance of the former species. These isolates could only be successfully grown in Novy-MacNeal-Nicolle + liver infusion-tryptose supplemented with 20-30% fetal calf serum. After 6 months of serial maintenance, rich and apparently pure cultures of T. rangeli were obtained. Both stocks were analyzed with different approaches and compared with two T. cruzi isolates also from chagasic patients under care at IPEC, besides T. rangeli and T. cruzi reference strains. All stocks were characterized by morphology, biometry, electrophoresis of isoenzymes, and products of kDNA minicircle amplified by polymerase chain reaction. The identification of T. rangeli was largely confirmed by all techniques. Taken together, these data represent the third report on T. rangeli in human hosts in Brazil.     

The role played by sympatric collared peccary (Tayassu tajacu), white-lipped peccary (Tayassu pecari), and feral pig (Sus scrofa) as maintenance hosts for Trypanosoma evansi and Trypanosoma cruzi in a sylvatic area of Brazil

The Brazilian Pantanal has been considered one of the richest and most diverse wetland ecosystems in the world. It is occupied by cattle ranching, and a variety of wildlife species share the same habitats with domestic livestock. We investigated infections of Trypanosoma evansi and Trypanosoma cruzi in the sympatric suiformes-collared peccary (Tayassu tajacu), white-lipped peccary (Tayassu pecari), and feral pig (Sus scrofa) by parasitological, serological, and molecular tests. Additionally, we evaluated the health status of both positive and negative suiformes by hematological and biochemical parameters. The results show that peccaries and feral pigs play an important role on the maintenance of both T. evansi and T. cruzi in the Brazilian Pantanal. Health impairment was observed only in the white-lipped peccary infected with T. evansi. Despite presenting low T. evansi parasitemia, all infected white-lipped peccaries displayed low hematocrit values and marked leucopenia. The hematological values showed that the T. evansi infection is more severe in young white-lipped peccaries. The presented data show that feral pigs and peccaries are immersed in the transmission net of both trypanosome species, T. cruzi and T. evansi, in the Pantanal region.     

Expression in Escherichia coli of a dominant immunogen of Trypanosoma cruzi recognized by human chagasic sera.

A genomic clone expressing a Trypanosoma cruzi antigen in Escherichia coli was identified using human chagasic sera. Chagasic antibodies affinity purified on extracts of this clone recognized a high-molecular-weight protein expressed in all developmental stages of the parasite life cycle, as well as in various T. cruzi strains. The antigen is associated with the cytoskeleton of the parasite and localizes along the attachment region between the flagellum and the cell body. Antibodies to the recombinant antigen were detected in the sera of 115 chagasic patients from different endemic regions, but not in sera of patients with leishmaniasis, T. rangeli infection, or other parasitic diseases. Our data suggest that the presence of antibodies to this antigen may be specifically associated with Chagas' disease.     

Interleukin-12-mediated resistance to Trypanosoma cruzi is dependent on tumor necrosis factor alpha and gamma interferon.

The aim of this study was to determine if interleukin-12 (IL-12) has a role in the immune response to Trypanosoma cruzi. Infection of BALB/c mice with the virulent Tulahuen strain of T. cruzi is characterized by a high-level parasitemia, pathology in the heart associated with the presence of amastigotes, and death during the acute phase of the disease. Administration of IL-12 to BALB/c mice infected with T. cruzi resulted in a reduced parasitemia and a significant delay in the time to death compared with those for infected controls. This protective effect was correlated with increased levels of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in serum. To determine if these cytokines were involved in the protective effects of IL-12, we treated infected mice with IL-12 alone or in combination with monoclonal antibodies specific for IFN-gamma or TNF-alpha. These antibodies antagonized the protective effect of exogenous IL-12. Treatment of infected mice with a polygonal antibody specific for IL-12 resulted in a significant increase in parasitemia but did not affect the time to death. These latter studies demonstrate a role for endogenous IL-12 in resistance to T. cruzi. Together, our data identify an IL-12-mediated mechanism of resistance to T. cruzi, which is dependent on IFN-gamma and TNF-alpha.     

Isolation of a Trypanosoma cruzi antigen by affinity chromatography with a monoclonal antibody. Preliminary evaluation of its possible applications in serological tests.

By affinity chromatography with a monoclonal antibody (163B6), obtained in our laboratory, we have isolated a T. cruzi antigen which could be useful for differential diagnosis of Chagas' disease from leishmaniasis. This antigen, a 52-kD protein, reacted with all sera from Chagas' disease patients tested but not with sera from patients with leishmania, in ELISA. The 52-kD antigen is widely distributed in the Trypanosoma genus since the 163B6 monoclonal antibody reacts with T. rangeli and 8 strains and a clone of T. cruzi epimastigotes.     

Interaction of Trypanosoma rangeli Tejera, 1920 with different cell lines in vitro

Intracellular multiplication of Trypanosoma rangeli was evaluated in vitro using experimental infection of Vero cells, murine macrophages, and promonocytes with T. rangeli Choachi, Macias, and SC-58 clone B1 strains. Our results revealed a low infectivity of all T. rangeli strains to these cell lines. Macrophages showed the highest infection rate; however, intracellular forms were no longer observed 48 h post infection Despite the observation of intracellular parasites up to 144 h post infection, the infection rates of Vero cells and J774G.8 promonocytes with these parasite strains were always below 5%. Pre-incubation of parasites with normal mouse serum increased the initial infectivity but not the time course of the infection. Under our experimental conditions, we did not observe any evidence of intracellular multiplication of T. rangeli within these cell lines.     

Primary structure of Trypanosoma cruzi small-subunit ribosomal RNA coding region: comparison with other trypanosomatids

A Trypanosoma cruzi small subunit ribosomal RNA gene was sequenced from genomic recombinant plasmid clones. The assigned coding region was 2319 bp, the longest SSU rRNA gene described to date. On the basis of comparisons with published sequences from Crithidia fasciculata, Trypanosoma brucei, and Leishmania donovani, we conclude that the extra nucleotides in the T. cruzi gene occur in highly variable regions of rRNA genes. A phylogenetic analysis was performed with the SSU rRNA sequences from these four trypanosomatids and from Euglena gracilis as an outgroup. The Leishmania and Crithidia sequences were remarkably similar to each other (not separable statistically). Given the standard errors associated with the branching order of the two Trypanosoma species and the Leishmania-Crithidia branch, the actual topology cannot be unequivocally determined using only the ribosomal sequences analyzed here.